CN109312325B - Novel cedar pollen protein - Google Patents
Novel cedar pollen protein Download PDFInfo
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- CN109312325B CN109312325B CN201780036197.6A CN201780036197A CN109312325B CN 109312325 B CN109312325 B CN 109312325B CN 201780036197 A CN201780036197 A CN 201780036197A CN 109312325 B CN109312325 B CN 109312325B
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Abstract
The present invention provides a novel cedar pollen protein and a diagnostic agent, a prophylactic agent, a therapeutic agent, and the like for allergic diseases caused by cedar pollen using the same. The cedar pollen protein is selected from the following (a) to (c): (a) a protein comprising the amino acid sequence represented by SEQ ID No. 2; (b) a protein having cedar pollen allergen activity, which comprises an amino acid sequence in which 1 or more amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO. 2; (c) a protein having cedar pollen allergen activity, which comprises an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO. 2.
Description
Technical Field
The present invention relates to a novel protein derived from cedar pollen and use of the protein.
Background
Pollinosis is an allergic disease which is caused by pollen flying in the air by inhalation and shows symptoms such as allergic conjunctivitis, rhinitis, inflammation of the skin, and asthma, such as itching and pain of the eyes. Many plants are known as causes of pollinosis, but the most frequent pollinosis in japan is cedar (スギ, cedar) pollinosis (an allergic disease caused by cedar pollen). In the report of 2003, the incidence of cedar pollinosis was estimated to be 19.4% in 2001 (non-patent document 1), and it was estimated that at least 2000 million people are afflicted with cedar pollinosis, and it was not known that the disease was called national disease.
One of the more serious problems associated with cedar pollinosis is the annual increase in the amount of cedar pollen scattered. It is known that the amount of fir pollen scattering is positively correlated with the average temperature in summer one year before the pollen scattering period, and it has been reported that the amount of fir pollen scattering increases year by year as the average temperature in japan increases (non-patent document 2). Accordingly, it is also easy to think that the number of patients with cedar pollinosis will increase in the future, which may damage the health of the Japanese. Actually, it has been reported that the incidence of cedar pollinosis has increased 2.6 times from 1980 to 2000, as a result of meta-analysis (meta-analysis) using 38 reports (non-patent document 3).
Among these treatments for cedar pollinosis, a series of symptomatic therapies represented by the use of antihistamines and the like have been used, and allergen immunotherapy (desensitization therapy) is expected as the only treatment method that can be expected to achieve radical cure of cedar pollinosis. This is a therapeutic method expected to alleviate allergic symptoms by administering an allergen causing an allergic disease subcutaneously or sublingually or the like. In the development of these allergen immunotherapy methods or improvement of the therapeutic effect thereof, it is essential to identify causative allergens and understand them.
As main cedar pollen allergens associated with the onset of cedar pollinosis, Cry j 1, Cry j 2, Cry j 3 (non-patent documents 4, 5, and 6), particularly Cry j 1 and Cry j 2, have been reported so far, and standardized cedar pollen extracts containing them as main components have been developed (non-patent document 7), and allergen immunotherapy for cedar pollinosis has been carried out in japan. However, there are also patient populations where allergen immunotherapy cannot be effective. It has been suggested that a novel allergen which is not identified is present in cedar pollen (non-patent document 8), and a novel allergen protein which is more effective and is characteristic for immunotherapy of cedar pollinosis is required.
Documents of the prior art
Patent document
Non-patent document 1: okuda M, Ann Allergy Ashma Immunol, 91:288-
Non-patent document 2: yamada T, et al, J Allergy Clin Immunol, 133:632-639,2014
Non-patent document 3: kaneko Y, et al, Int Arch Allergy immunol.,136:365-
Non-patent document 4: yasueda H, et al, J.allergy Clin.Immunol.,71:77-86,1983
Non-patent document 5: sakaguchi M, et al, Allergy,45:309-
Non-patent document 6: fujimura T, et al, Allergy,62:547-553,2007
Non-patent document 7: yasueda H, et al, Allergol. Int.,46:135-140,1997
Non-patent document 8: fujimura T, et al, Int Arch Allergy, 133:125-
Disclosure of Invention
Problems to be solved by the invention
The present invention provides a novel cedar pollen protein and a diagnostic agent, a prophylactic agent, a therapeutic agent, and the like for allergic diseases caused by cedar pollen using the same.
Means for solving the problems
The present inventors have made studies to solve the above-mentioned problems, and have found that a novel protein exhibiting high reactivity with IgE in lymphocytes and serum from patients with cedar pollenosis is found from a cedar pollen crude antigen, and that the protein is effective as a diagnostic agent, a prophylactic agent or a therapeutic agent for allergic diseases caused by cedar pollens.
That is, the present invention relates to the following 1) to 13).
1) A cedar pollen protein selected from the following (a) to (c).
(a) A protein comprising the amino acid sequence represented by SEQ ID No. 2;
(b) a protein having cedar pollen allergen activity, which comprises an amino acid sequence in which 1 or more amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO. 2;
(c) a protein having cedar pollen allergen activity, which comprises an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO. 2.
2) A polynucleotide encoding a cedar pollen protein selected from the group consisting of (a) to (c) below.
(a) A protein comprising the amino acid sequence represented by SEQ ID No. 2;
(b) a protein having cedar pollen allergen activity, which comprises an amino acid sequence in which 1 or more amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO. 2;
(c) a protein having cedar pollen allergen activity, which comprises an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO. 2.
3) A polynucleotide selected from the following (d) to (f).
(d) A polynucleotide comprising the base sequence represented by SEQ ID NO. 1;
(e) a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO. 1 and encodes a protein having cedar pollen allergenic activity;
(f) a polynucleotide encoding a protein having cedar pollen allergen activity, the polynucleotide comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO. 1.
4) An agent for preventing or treating allergic diseases caused by cedar pollen, which comprises the cedar pollen protein of 1) as an active ingredient.
5) A diagnostic agent for allergic diseases caused by cedar pollen, containing the cedar pollen protein of 1) as effective component.
6) An antibody against the cedar pollen protein of 1) above.
7) The antibody against the cedar pollen protein according to 6) above, which is a monoclonal antibody.
8) Use of the cedar pollen protein of 1) above for producing a prophylactic or therapeutic agent for allergic diseases caused by cedar pollen.
9) Use of the cedar pollen protein of 1) above in the manufacture of a diagnostic agent for allergic diseases caused by cedar pollen.
10) The cedar pollen protein according to 1) above, which is used for preventing or treating allergic diseases caused by cedar pollen.
11) The cedar pollen protein according to 1) above, which is used for diagnosing allergic diseases caused by cedar pollen.
12) A method for preventing or treating an allergic disease caused by cedar pollen, which comprises administering the cedar pollen protein of 1) above to a patient.
13) A method for diagnosing an allergic disease caused by cedar pollen, comprising administering the cedar pollen protein of 1) above to a patient.
Effects of the invention
The cedar pollen protein of the present invention can be used as a diagnostic agent, a prophylactic agent, a therapeutic agent, and the like for allergic diseases caused by cedar pollen. The cedar pollen protein contains amino acid primary sequences which are not identical with Cry j 1, Cry j 2 and other known cedar pollen proteins, so that the combination of the cedar pollen protein and the known cedar pollen protein can perform more effective desensitization treatment.
Drawings
FIG. 1 is a diagram showing the analysis of purified recombinant cedar pollen protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
FIG. 2 shows the results of measurement of proliferation reaction caused by addition of purified recombinant protein using peripheral blood mononuclear cells of 4 patients with cedar pollinosis (ImmunoCAP Score. gtoreq.2).
FIG. 3 is a graph showing the results of flow cytometry, which shows that basophils in peripheral blood of 13 patients with cedar pollinosis (ImmunoCAP Score. gtoreq.2) were activated by purified recombinant protein to enhance the expression of CD203c, while the expression of CD203c was not changed in basophils in peripheral blood of 2 healthy subjects (ImmunoCAP Score. gtoreq.2).
Detailed Description
Cunninghamia lanceolata pollen protein
The cedar pollen protein of the present invention is a protein selected from the following (a) to (c).
(a) A protein comprising the amino acid sequence represented by SEQ ID No. 2;
(b) a protein having cedar pollen allergen activity, which comprises an amino acid sequence in which 1 or more amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO. 2;
(c) a protein having cedar pollen allergen activity, which comprises an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO. 2.
The cedar pollen protein of the present invention includes a protein comprising an amino acid sequence in which 1 or more amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO. 2, as long as it has cedar pollen allergen activity (the above (b)). Examples of the cedar pollen protein containing such an amino acid sequence include a variant of the cedar pollen protein having the amino acid sequence shown in SEQ ID NO. 2.
The plurality of amino acids to be deleted, substituted or added is, for example, 1 to 10, more preferably 1 to 5 amino acids. The addition or deletion includes addition or deletion of 1 to several amino acids at both ends.
Specific examples of the isoforms include deletion of 4 residues at the C-terminus.
Among them, the "cedar pollen allergen activity" includes not only the activity of binding to IgE on mast cells to cause immediate type allergic reaction to atopic persons (De Weck, AL.et al., int. Arch. allergy immunol.,146:177-189,2008), but also the activity of binding only to IgE in serum. The IgE binding activity can be measured by the method described in International publication pamphlet 2012/105541, etc.
The cedar pollen protein of the present invention includes a protein that contains a protein having 90% or more identity to the amino acid sequence shown in sequence No. 2 when the corresponding sequence is appropriately aligned with the amino acid sequence shown in sequence No. 2, as long as it has cedar pollen allergen activity ((c) above).
Among them, the identity with the amino acid sequence represented by SEQ ID NO. 2 is preferably 95% or more, and more preferably 98% or more. For the identity of the amino acid sequence, for example, a method using BLAST (basic Local Alignment Search Tool) for the National Center for Biological information, and optional parameters for calculation using initial settings can be applied.
Furthermore, the cedar pollen protein of the present invention may be a fusion with a protein or the like to form a sequence such as a multiple histidine residue which is useful for purification and to ensure stability in recombinant production.
Polynucleotides encoding fir pollen proteins
The polynucleotide of the present invention is a polynucleotide encoding the above-mentioned cedar pollen protein, and preferably includes (d) a polynucleotide comprising the base sequence represented by SEQ ID NO. 1; (e) a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO. 1 and encodes a protein having cedar pollen allergenic activity; (f) a polynucleotide encoding a protein having cedar pollen allergen activity, the polynucleotide comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO. 1.
(e) The polynucleotides of (a) and (f) comprise variants of the polynucleotides of (d). Such variants include natural adversary variants or naturally-occurring variants that can be generated using mutagenesis techniques well known in the art.
The polynucleotide of the present invention includes not only double-stranded DNA but also various single-stranded DNAs or RNAs such as sense strand and antisense strand constituting the double-stranded DNA. The antisense strand can be used as a probe or the like. The DNA includes, for example, cDNA or genomic DNA obtained by cloning or chemical synthesis techniques or a combination thereof. The polynucleotide of the present invention may be added with a nucleotide sequence such as a sequence of an untranslated region (UTR) or a vector sequence (including an expression vector sequence) in addition to a nucleotide sequence encoding the polypeptide of the present invention.
The stringent conditions include, for example, the conditions described in Molecular Cloning, A Laboratory Manual (Second Edition, J.Sambrook et al, 1989). That is, hybridization is carried out under conditions in which a solution containing 6 XSSC (1 XSSC composition: 0.15M sodium chloride, 0.015M sodium citrate, pH7.0), 0.5% SDS, 5 XDenhardt and 100mg/mL herring sperm DNA is incubated with a probe at 65 ℃ for 8 to 16 hours.
The identity with the base sequence represented by SEQ ID NO. 1 is preferably 95% or more, more preferably 98% or more. For the identity of the nucleotide sequence, a method of calculating the initial set value using BLAST and optional parameters can be applied.
Examples of the polynucleotide variants include those having a mutation at position 4, a mutation at position 898, a mutation at position 1029, and a mutation at position 1643 in the base sequence shown in SEQ ID NO. 1, wherein a is a mutation, g is a mutation, t is a mutation, and t is a mutation.
Obtaining of polynucleotide encoding pollen protein of Cunninghamia lanceolata
The polynucleotide encoding a cedar pollen protein of the present invention can be cloned from cedar pollen, and examples of cloning methods include methods using known methods, for example, the shotgun method and the PCR method.
For example, a probe that specifically hybridizes to a part of the nucleotide sequence of the polynucleotide of the present invention may be prepared, and a genomic DNA library or a cDNA library may be screened using the probe. Any sequence and length may be used as the probe as long as it specifically hybridizes to at least a part of the polynucleotide of the present invention or the complementary strand thereof. Further, a method of artificially synthesizing a polynucleotide can be exemplified. (Kosuri S et al, Nature Methods 11, 499-one 507,2014)
The polynucleotide of the present invention can also be obtained by obtaining a sequence that hybridizes to a polynucleotide including a part or all of the polynucleotide of the present invention using an appropriate principle. Examples thereof include a PCR method using the above-mentioned polynucleotide comprising a part of the polynucleotide of the present invention as a primer, and a method using the above-mentioned polynucleotide comprising a part of the polynucleotide of the present invention as a probe.
For example, in a method using an amplification method such as PCR, a large number of DNA fragments including the polynucleotide can be obtained by preparing primers from sequences on the 5 'side and the 3' side of the polynucleotide of the present invention (or complementary sequences thereof), respectively, performing an amplification reaction such as PCR using these primers and using genomic DNA (or cDNA) or the like as a template, and amplifying a DNA region sandwiched between the primers.
The polynucleotide provided by the present invention can also be produced by altering a polynucleotide comprising the base sequence represented by SEQ ID NO. 1 by a method such as a planned or random mutagenesis method.
Among them, planning to introduce a mutation at the time of mutation can be performed, for example, by referring to a characteristic sequence on a polynucleotide sequence. Examples of the method for randomly introducing mutations include a PCR method and a method using a mutagen treatment. A method for introducing mutation in a planned manner includes a Site-Directed Mutagenesis method, and more specifically, it can be carried out using, for example, a Site-Directed Mutagenesis System Mutan-Super Express Km kit (Takara Bio). In addition, a recombinant PCR method (PCR protocols, Academic Press, New York,1990) may also be used.
Preparation method of cedar pollen protein
The cedar pollen protein of the present invention can be obtained by separating and refining cedar pollen. The method of separation and purification is not particularly limited, and the cedar pollen extract can be separated and purified by a conventionally known method such as gel filtration, ion exchange chromatography, affinity chromatography, or the like.
A recombinant vector prepared by inserting the polynucleotide of the present invention into an appropriate vector may be introduced into a host cell, and the cedar pollen protein may be expressed intracellularly or extracellularly and collected.
The host is not particularly limited as long as it is a viable cell that can be transformed, and examples thereof include bacteria such as Escherichia coli and Bacillus subtilis, fungi such as yeast and filamentous fungi, insect culture cells such as Sf9 cells, insects such as silkworms, animal cells, plants, and plant-derived cells.
The vector into which the polynucleotide of the present invention is inserted is not particularly limited as long as it can replicate in the above-mentioned host, and may be appropriately determined depending on the type of host to be introduced, the method of introduction, and the like.
Examples thereof include plasmid DNA, phage DNA, and viral vectors. The vector DNA used for the construction of the expression vector can be any of the widely available vectors. Examples thereof include pUC19(Takara Bio), pTV118N (Takara Bio), pMAMneo (Clontech), pGEX (GE healthcare), pET160(Invitrogen), pDEST (Invitrogen), pEx (Merckmill), pBacPAK (Clontech), and the like. Examples of the viral vector include DNA viruses and RNA viruses such as baculovirus vectors, retrovirus vectors, and Human Immunodeficiency Virus (HIV) lentivectors, adenovirus vectors, adeno-associated virus vectors (AAV vectors), herpes viruses, vaccinia viruses, poxviruses, poliovirus, Sindbis (Sindbis) viruses, sendai viruses, and monkey virus-40 (SV-40).
When a host is transformed with the recombinant vector, the transformation can be performed by a protoplast method, a competent cell method, an electroporation method, or the like. The obtained transformant can be cultured under appropriate conditions using a medium containing a carbon source, a nitrogen source, a metal salt, vitamins, and the like, which can be utilized. The above-obtained culture solution can be used to collect and purify proteins by a general method, thereby obtaining the cedar pollen protein of the present invention. For example, when Escherichia coli is used as the host, the method described in pET System Manual,10th edition (Novagen) and the like can be exemplified.
Agent for preventing or treating allergic diseases caused by cedar pollen
The cedar pollen protein of the present invention can be used as a prophylactic or therapeutic agent for allergic diseases caused by cedar pollen, and is administered to a person (patient) in need thereof to prevent or treat allergic diseases caused by cedar pollen.
Among them, examples of allergic diseases caused by cedar pollen include all allergic diseases caused by antigens specific to cedar pollen, and specifically, for example, atopic bronchial asthma, allergic rhinitis, allergic conjunctivitis, and atopic dermatitis.
Such a preventive or therapeutic agent for an allergic disease caused by cedar pollen can be used, for example, as a desensitizing therapeutic agent for an allergic disease caused by cedar pollen.
Regarding the cedar pollen protein of the present invention, according to the results of amino acid identity analysis using GENETYX ver.12, no identity was found with Cry j 1, Cry j 2, Cry j 3 reported as the main cedar pollen allergen associated with the development of cedar pollinosis, and it is considered that since it is a different protein from presently known Cry j 1, Cry j 2, Cry j 3, by combining them, more effective desensitization treatment can be performed.
When the agent for preventing or treating allergic diseases caused by cedar pollen of the present invention is used as a desensitizing agent, it is preferable to use the cedar pollen protein of the present invention as it is, or dry it to a powder, or add, if necessary, usual adjuvants and various additives such as stabilizers, excipients, dissolution aids, opacifying agents, buffers, painless agents, preservatives, colorants, and the like by a usual method to prepare a compounding agent.
For example, purified cedar pollen protein in powder form can be dissolved in physiological saline to which phenol is added and used as a stock solution of an antigen for desensitization therapy.
The agent for preventing or treating allergic diseases caused by cedar pollen according to the present invention can be administered by a common administration route, for example, transdermal, oral, intradermal, subcutaneous, intramuscular, intraperitoneal or the like. Further, the composition can be used as a transdermal or transmucosal drug such as a buccal, sublingual tablet, eye drop, nasal spray, paste, emulsion, or lotion.
The dose and the number of administrations of the agent for preventing or treating allergic diseases caused by cedar pollen of the present invention vary depending on the administration route, symptoms, and the like, and are appropriately selected, for example, in the range of about 0.1 to 1000 μ g per 1 adult, and about 1 to several times per week.
Diagnosis of allergic diseases caused by cedar pollen
The cedar pollen protein can form an allergic disease diagnostic drug caused by cedar pollen and is used for diagnosing allergic diseases caused by cedar pollen.
Such a diagnostic agent for allergic diseases caused by cedar pollen can be used as a diagnostic agent for skin reactions against allergic diseases caused by cedar pollen, i.e., an intradermal test or prick test agent. Furthermore, by preparing a specific IgE antibody test drug, diagnosis using serum or plasma of a patient can be performed. (nasal allergy diagnosis and treatment guide-perennial rhinitis and pollinosis-2013 edition, Life Sciences of Kabushiki Kaisha)
When used as a diagnostic agent for skin reactions, the cedar pollen protein of the present invention obtained by the above-mentioned method may be dried, for example, to a powder form, dissolved in physiological saline containing phenol or glycerol, and diluted for use. Examples of the specific IgE antibody test reagent include a reagent in which an IgE antibody in a patient sample is bound to the cedar pollen protein of the present invention in an aqueous phase or a solid phase, and detection is performed based on the principle of a fluorescence enzyme immunoassay (fluoroenzyme immunoassay), a Chemiluminescence enzyme immunoassay (chemiluminescent enzyme immunoassay), an enzyme immunoassay (enzyme immunoassay), or the like, but the detection method is not limited thereto.
Antibody of cedar pollen protein
The antibody against the cedar pollen protein of the present invention is an antibody capable of specifically binding to the cedar pollen protein of the present invention. The antibody refers to an immunoglobulin (IgA, IgD, IgE, IgG, IgM, and Fab fragments thereof, F (ab')2 fragments, Fc fragments), and examples thereof include a polyclonal antibody, a monoclonal antibody, a single chain antibody, an anti-idiotype antibody (anti-idiotype antibody), and a humanized antibody, but are not limited thereto.
The antibody can be produced by various known methods, and the production method is not particularly limited.
The above-mentioned antibody can be used for identification of an organism, a tissue or a cell thereof expressing the cedar pollen protein of the present invention. For example, it can be used for measuring the presence or absence of cedar pollen proteins in the atmosphere, in indoor spaces, or on human mucous membranes. The measurement can be carried out by a known immunological method, for example, by ELISA.
The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
Examples
Example 1 crude purification of novel cedar pollen protein
< crude purification of cedar pollen protein >
50mL of extraction buffer (20mM Tris-HCl, pH9.0) was added to 2g of cedar pollen, and ultrasonication was performed for 10 minutes. Subsequently, the mixture was mixed at room temperature for 10 minutes and centrifuged (10,000g, 10 minutes) to obtain a supernatant. The supernatant was again subjected to ultracentrifugation (50,000g, 60 minutes), and the supernatant was recovered. To 17mL of the supernatant, an equal amount of a Q sepharose high performance resin (GE healthcare) suspension equilibrated with an extraction buffer (20mM Tris-HCl, pH9.0) was added, and the mixture was shaken at room temperature for 30 minutes. After removing the supernatant by centrifugation (1,000g, 10 minutes), the volume was adjusted to 50mL with an extraction buffer (20mM Tris-HCl, pH9.0), the mixture was inverted, and the resin was washed by centrifugation again. Next, 10mL of an extraction buffer (20mM Tris-HCl, pH9.0) containing 0.0125M NaCl was added to the washed resin, and the mixture was shaken at room temperature for 5 minutes. The supernatant was removed by centrifugation (1,000g, 10 minutes), and the protein adsorbed to the resin was eluted. The same operation was repeated using an extraction buffer (20mM Tris-HCl, pH9.0) containing 0.025M NaCl, an extraction buffer (20mM Tris-HCl, pH9.0) containing 0.05M NaCl, a draw buffer (20mM Tris-HCl, pH9.0) containing 0.1M NaCl, an extraction buffer (20mM Tris-HCl, pH9.0) containing 0.2M NaCl, an extraction buffer (20mM Tris-HCl, pH9.0) containing 0.4M NaCl, and an extraction buffer (20mM Tris-HCl, pH9.0) containing 0.6M NaCl. Finally, the eluted protein solution was recovered as a crude purified solution using an extraction buffer (20mM Tris-HCl, pH9.0) containing 0.6M NaCl.
< identification of partial internal amino acid sequence >
The recovered crude refined solution was subjected to a concentration operation using a 100,000MWCO spin column. The concentrated sample was subjected to reduction and SDS-PAGE using a general protocol of Novex (registered trademark) NuPAGE (registered trademark) SDS-PAGE system (invitrogen). The SDS-PAGE gel after electrophoresis was Silver stained using the standard protocol of the Silver Staining Kit (registered trademark). Next, in order to clarify a partial amino acid sequence in the protein of the present invention, a fragment obtained by protease restriction decomposition is analyzed. That is, after the gel having a molecular weight of 70kDa in the vicinity of the band was minced, the fragment was collected, washed with water and acetonitrile, and subjected to alkylation treatment with DTT and iodoacetamide. Next, trypsin was added to the sample, and the mixture was incubated at 37 ℃ for 16 hours to carry out hydrolysis. After the reaction, peptides were extracted from the gel pieces using 25mM ammonium bicarbonate, 5% formic acid and acetonitrile. Finally, the extraction solution was dried under reduced pressure. The dried sample was dissolved in a solvent (volume ratio 98:2:0.1) containing water, acetonitrile and trifluoroacetic acid, and analyzed by an LC-MS/MS system (Paradigm MS4(Michrom biolesources corporation), MS/MS: LTQ OrbiTrap XL mass analyzer (ThermoFisher).) Column selection analysis C18 Column L-Column Micro (chemical evaluation research institution), mobile phase a ([ water ]: acetonitrile ]: formic acid ═ 98:2:0.1 (volume ratio)) and mobile phase B ([ water ]: acetonitrile ]: 5:95:0.1 (volume ratio)). after the sample was added, the molecular weight was measured by an acetonitrile liquid-feeding gradient (minute,% B) → (0, 5) → (20, 35) → (25, 90) → (30, 90) → (30.01, 5) → (40, 5) → (MS, 5) → molecular weight fractionation by using conditions of elution and molecular weight fractionation, peptide sequences were determined using mascot (matrix science) software. As a result, the partial sequence WIVDEATGLR was obtained.
EXAMPLE 2 preparation of purified recombinant protein
< extraction of Total RNA of Cunninghamia lanceolata >
2g of the pulverized male flowers (anthers) of Cunninghamia lanceolata frozen in liquid nitrogen was added to a 50mL tube containing 25mL of a Plant RNA Isolation Reagent (Invitrogen), mixed, and allowed to stand at room temperature for 5 minutes.
After centrifugation at 4 ℃ (2600x g, 5 minutes), the upper layer was filtered by decantation through a mesh with a mesh size of 100 μm. Subsequently, 1/5-fold amount of 5M NaCl and 3/5-fold amount of chloroform were added to the recovered filtrate. After centrifugation at 4 ℃ (2600x g, 30 minutes), 20mL of the upper layer was recovered, and 9/10 times the amount of isopropanol was added thereto and stirred. After standing at room temperature for 10 minutes, the resulting mixture was centrifuged at 4 ℃ (2600x g for 30 minutes) to precipitate RNA. After removing the supernatant, 10mL of 75% ethanol was added to the RNA pellet and washed. Then, after centrifugation at 4 ℃ for 5 minutes (2600x g), the supernatant was removed, and Total RNA was dried at room temperature and then dissolved in 200. mu.L of RNase free water. After centrifugation at room temperature (12000x g, 3 minutes), the cells were treated with RNase-Free DNase Set (QIAGEN) for DNase. That is, 15. mu.L of Buffer RDD and 5. mu.L of DNase I stock solution were added to 180. mu.L of the supernatant of the Total RNA solution, and the mixture was allowed to stand at room temperature for 10 minutes. Next, total RNA was purified using RNeasy mini kit (QIAGEN). That is, 700. mu.L of Buffer RLT and 500. mu.L of ethanol were added to 200. mu.L of the sample after DNase treatment, and then the mixture was added to a column attached to the RNeasy mini kit to perform centrifugation (8000x g, 15 seconds).
Subsequently, 500. mu.L of Buffer RPE was added to the column, centrifuged at room temperature (8000x g, 15 seconds), and the operation was repeated 2 times. Next, 30. mu.L of RNase free water was added to the column, and centrifuged at room temperature (8000x g, 1 minute), and the resulting solution was collected as total RNA after purification.
< preparation of cDNA of Cunninghamia lanceolata >
cDNA was synthesized from the resulting purified total RNA using Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen). Mu.g of Total RNA2 was dissolved in 8. mu.L of purified water, and 1. mu.L of 50. mu.M oligo (dT)20 and 1. mu.L of 10mM dNTP were added thereto, followed by incubation at 65 ℃ for 5 minutes. After cooling the samples on ice, 2. mu.L of 10 RT buffer, 4. mu.L of 25mM MgCl2, 2. mu.L of 0.1M DTT, 1. mu.L of RNaseOUT (40U/. mu.L), 1. mu.L of SuperScript III Reverse Transcriptase were added and mixed. After incubation at 50 ℃ for 50 minutes, the reaction was stopped by treatment at 85 ℃ for 5 minutes. Next, 1. mu.L of RNase H was added to the sample and incubated at 37 ℃ for 20 minutes, thereby forming a fir cDNA.
< clone >
The partial sequence WIVDEATGLR thus obtained was deduced into cDNA sequence using GENETYX Ver.12.0.4 software, and identity search was performed using ForestGEN (http:// ForestGEN. ffpri. affrc. go. jp/ja/index. html) database. As a result, the cluster ID Cj5792 was searched. It was also found that the unidentified protein gene sequence gi |157358255| emb | CAO65892.1| from grape (Vitis vinifera) as a homologous gene to the retrieved Cj5796 comprises ForestGEN cluster ID: Cj 822. Furthermore, Cj5792 and Cj822 have high identity with the fir cDNA libraries BY882225.1 and BY900252.1, respectively. Based on this information, a primer set (primer set) for cloning was designed for the following study. That is, nested PCR (nested PCR) was performed using KOD Fx Neo (TOYOBO) using the cDNA of Taxus chinensis as a template, Primer 1 (GATTTCATGRCAGCGACAG: SEQ ID NO. 3) and Primer 2 (CATGRCAGCGACAGSGAT: SEQ ID NO. 4) downstream thereof as sense primers and Primer 3 (TCAAAGTTGATGCAACAATTG: SEQ ID NO. 5) as antisense primers. The PCR product was purified by Wizard SV Gel and PCR Clean-Up System (Promega). The purified PCR product was inserted into pCR 2.1-TOPO TA Vector using TOPO TA Cloning Kit for Sequencing (Thermo Fischer) Kit to prepare a Vector containing the polynucleotide represented by SEQ ID NO. 1.
< purification of recombinant protein >
A recombinant protein having a Flag tag added to the C-terminus of the primary amino acid sequence of SEQ ID NO. 2 (excluding the amino acids at positions 1 to 26 of the presumed signal sequence portion) was produced by the method of Japanese patent application laid-open No. 2014-195411 using the ProCube (registered trademark) service of Sysmex corporation. That is, using the vector prepared as described above, the polynucleotide represented by SEQ ID No. 1 (except for the polynucleotides at positions 1 to 78 encoding amino acids 1 to 26 in the presumed signal sequence portion) was recombined with a transfer plasmid having a signal sequence for silkworm expression and a Flag tag fusion-expressed at the C-terminus to prepare a baculovirus, which was then inoculated into silkworm pupae, followed by trituration of the pupae obtained on day 7 using a trituration buffer (20mM Trs-HCl, 100mM NaCl, Complete EDTA free 1 pellet/50 mL, phenylthiourea). After ultracentrifugation of the ground product, the supernatant was collected and subjected to affinity PURIFICATION using DDDDDDK-tagged Protein PURIFICATION GEL (MBL) according to the attached file, thereby producing a purified recombinant Protein. The results of the analysis of the purified recombinant protein by SDS-PAGE are shown in FIG. 1.
Example 3 determination of allergen Activity of purified recombinant protein
(i) Lymphoball proliferative response in patients with cedar pollinosis
Peripheral blood mononuclear cells were separated from 30mL of peripheral blood obtained from patients with Cunninghamia lanceolata (ImmunoCAP score. gtoreq.2) to yield a cell size of 1.11X 106cells/mL were suspended in RPMI-1640 medium containing 10% non-activated autologous plasma. The prepared cell suspension 180. mu.L and 20. mu.L of the recombinant protein solution of the present invention (concentration 100. mu.g/mL) were inoculated into a 96-well plate (2X 10)5cells/well). At 37 deg.C, 5% CO2After culturing under the conditions for 3 days, adding3H-thymidine was measured as radioactivity at 16 hours, and cell proliferation was evaluated. Calculating when adding purified recombinant protein3The Stimulation index (Stimulation index) was obtained as the value obtained by dividing the intake of H-thymidine by the intake of H-thymidine in the absence of addition, and the result is shown in FIG. 2. In general, Stimulation index of 1.8 or more is used as a positive standard in a lymphocyte Stimulation test in clinical examination (Netherlands' repeat et al, liver volume 30, No. 4 439-443, 1989). From this criterion, 3 of 4 cases (75%) were judged to be positive. The above results show that the recombinant protein of the present invention is sensitized at a high frequency at the T cell level in patients with cedar pollinosis.
(ii) Activation of basophilic granulocyte of fir pollinosis patient
It is known that CD203c expression is enhanced in basophils after activation by allergen stimulation (De Weck, AL.et al, int. Arch. allergy immunol.,146:177-189, 2008). Based on this principle, the activation of basophils in blood of patients with cedar pollinosis (ImmunoCAP score ≧ 2) or healthy subjects (ImmunoCAP score <2) was analyzed using allergen (Allergenicity) kit (Beckman Coulter). That is, 20. mu.L of the recombinant protein solution of the present invention was added to 100. mu.L of whole blood obtained by heparin blood collection so that the final concentration became 3. mu.g/mL, and 20. mu.L of PBS (-) was added to the negative control sample. Next, 20. mu.L of an antibody mixture containing CD3-PC7, CRTH2-FITC and CD203c-PE was added to all the samples, and incubated at 37 ℃ for 15 minutes. mu.L of the reaction-stopping solution and 2mL of the hemolytic fixing solution were added, and the reaction was performed at room temperature for 10 minutes. After centrifugation (200x g, 5 minutes), the supernatant was removed, 3mL of Phosphate Buffer (PBS) was added, and the mixture was centrifuged again. After removing the supernatant, the cells were suspended in PBS containing 0.1% formaldehyde and measured by flow cytometry (facscan, BD Biosciences). From the obtained data, basophils in blood cells were selected using CD3-PC7 negative and CRTH2-FITC positive as indicators, and the expression intensity of CD203c was analyzed. The data show the ratio (%) of CD203c positive basophils in the protein solution-stimulated sample of the present invention to the ratio (%) of CD203c positive basophils in each negative control sample, and values of 1.6 or more, which are 2 times the maximum value of 0.8% in healthy subjects, were determined to be positive. As a result, as shown in fig. 3, in 2 healthy subjects, no enhancement of expression of CD203c was observed in 2 cases, while in 13 cases (54%) of patients with cedar pollinosis, enhancement of expression of CD203c was observed in 7 cases (54%) among the patients with cedar pollinosis, and the difference was significant (p <0.05, Welch's t test). Therefore, it was shown that the patients with cedar pollinosis have IgE bound to the recombinant protein of the present invention at a high frequency, and that the recombinant protein of the present invention has allergen activity.
Sequence listing
<110> Dapeng chemical industry Co Ltd
<120> novel cedar pollen protein
<130> TH0107
<150> JP 2016-122318
<151> 2016-06-21
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 1659
<212> DNA
<213> Artificial sequence
<220>
<223> recombinant cedar pollen protein
<220>
<221> CDS
<222> (1)..(1659)
<400> 1
atg aca gcg aca gcg atg aca acg gcg gcg cta cga tta ctc ata gcg 48
Met Thr Ala Thr Ala Met Thr Thr Ala Ala Leu Arg Leu Leu Ile Ala
1 5 10 15
ttg ctt ctt gtg gcg gcg ccg gca gag tgc ctg ccg cta cgg gca cgt 96
Leu Leu Leu Val Ala Ala Pro Ala Glu Cys Leu Pro Leu Arg Ala Arg
20 25 30
ggc aga tgg atc gtg gac gag gca acg ggg ctg aga gta aag cta gca 144
Gly Arg Trp Ile Val Asp Glu Ala Thr Gly Leu Arg Val Lys Leu Ala
35 40 45
tgc gtc aat tgg ccg ggc cac ctg gag ccc ggg tta ccg gaa ggt ctc 192
Cys Val Asn Trp Pro Gly His Leu Glu Pro Gly Leu Pro Glu Gly Leu
50 55 60
aac cgg ttg ccg gtt acc aca ata gcg cac acc atc agc tct ctg ggc 240
Asn Arg Leu Pro Val Thr Thr Ile Ala His Thr Ile Ser Ser Leu Gly
65 70 75 80
ttc aat tgt gtc cgc ctc acc tac tcc att cag atg gtc acg gag aag 288
Phe Asn Cys Val Arg Leu Thr Tyr Ser Ile Gln Met Val Thr Glu Lys
85 90 95
agc tac aca gag gcc acg gtt ggg cag act ttt gcg cag cta aat ctg 336
Ser Tyr Thr Glu Ala Thr Val Gly Gln Thr Phe Ala Gln Leu Asn Leu
100 105 110
aca gaa ccc gcc tcg gga atc gag ggc aac aat ccg ggg ttc ctc caa 384
Thr Glu Pro Ala Ser Gly Ile Glu Gly Asn Asn Pro Gly Phe Leu Gln
115 120 125
ttg ggc cac gtg gct gca tac gat agc atc gtg gcg gca ctt gcg gag 432
Leu Gly His Val Ala Ala Tyr Asp Ser Ile Val Ala Ala Leu Ala Glu
130 135 140
gcg ggt gtc atg gtc atc ctg gac aac cat gtc agt aag ccc aag tgg 480
Ala Gly Val Met Val Ile Leu Asp Asn His Val Ser Lys Pro Lys Trp
145 150 155 160
tgc tgt gct gtg gat gat ggc aat ggg ttt ttc ggt gac agt tac ttc 528
Cys Cys Ala Val Asp Asp Gly Asn Gly Phe Phe Gly Asp Ser Tyr Phe
165 170 175
gac cct aga ttg tgg caa cga ggg cta ggt cta atg gct aca cat ttc 576
Asp Pro Arg Leu Trp Gln Arg Gly Leu Gly Leu Met Ala Thr His Phe
180 185 190
aat aac acg ccc aac gtc gtc gcc atg tcg ctt cgc aac gag cta cgt 624
Asn Asn Thr Pro Asn Val Val Ala Met Ser Leu Arg Asn Glu Leu Arg
195 200 205
ggc aac cga tcg acc tcg gca cgg tgg tca aag tac atg cag cgg ggt 672
Gly Asn Arg Ser Thr Ser Ala Arg Trp Ser Lys Tyr Met Gln Arg Gly
210 215 220
gcg gct acc gtc cac gag gcc aac cca aat gtc ctc gtt gtc ctc tcg 720
Ala Ala Thr Val His Glu Ala Asn Pro Asn Val Leu Val Val Leu Ser
225 230 235 240
ggg cta cac ttc gac acc atc ctc agc ttc tta ccg gtc cta cca gtt 768
Gly Leu His Phe Asp Thr Ile Leu Ser Phe Leu Pro Val Leu Pro Val
245 250 255
act tta cct ttt aaa gag aaa att gtg tat gaa ggg cac tgg tac tcc 816
Thr Leu Pro Phe Lys Glu Lys Ile Val Tyr Glu Gly His Trp Tyr Ser
260 265 270
ttc ggt gtg ccc tgg cat gat ggc ttg cct aat gac att tgt ttg aat 864
Phe Gly Val Pro Trp His Asp Gly Leu Pro Asn Asp Ile Cys Leu Asn
275 280 285
gag acg tcg cgg ttt aag gat aat att ggg ttt ttg acc tcg tcg att 912
Glu Thr Ser Arg Phe Lys Asp Asn Ile Gly Phe Leu Thr Ser Ser Ile
290 295 300
aat ggt act gcg gca cca ctt ttt gtt agc gag ttc gga att gat cag 960
Asn Gly Thr Ala Ala Pro Leu Phe Val Ser Glu Phe Gly Ile Asp Gln
305 310 315 320
aga tac gtg aat gat aat gat aac agg tat ttg aac tgt atc ttg gct 1008
Arg Tyr Val Asn Asp Asn Asp Asn Arg Tyr Leu Asn Cys Ile Leu Ala
325 330 335
ttt ctg gcg gag gaa gac gtg gac tgg gcg ctg tgg act atg ggg ggg 1056
Phe Leu Ala Glu Glu Asp Val Asp Trp Ala Leu Trp Thr Met Gly Gly
340 345 350
agc tat aat tac cgg tcg gac aag gag ccc gtt cag gac ttt gag gag 1104
Ser Tyr Asn Tyr Arg Ser Asp Lys Glu Pro Val Gln Asp Phe Glu Glu
355 360 365
acc tac ggc ttt ttc aat cgt gac tgg agt cgc atc agg aat cct gac 1152
Thr Tyr Gly Phe Phe Asn Arg Asp Trp Ser Arg Ile Arg Asn Pro Asp
370 375 380
ttt att tct cgc ctt aag gaa att caa cag ccc att caa gac cct tac 1200
Phe Ile Ser Arg Leu Lys Glu Ile Gln Gln Pro Ile Gln Asp Pro Tyr
385 390 395 400
tta tct cca ggg cca tat tat caa ata atc tac cac cct gca tca ggc 1248
Leu Ser Pro Gly Pro Tyr Tyr Gln Ile Ile Tyr His Pro Ala Ser Gly
405 410 415
ctc tgt gtt gaa tcc agc att gga aac acc att cat cta gga tca tgc 1296
Leu Cys Val Glu Ser Ser Ile Gly Asn Thr Ile His Leu Gly Ser Cys
420 425 430
cag agt gtg aga agc aga tgg aac tat gat gcc agt gtg gaa ggc cca 1344
Gln Ser Val Arg Ser Arg Trp Asn Tyr Asp Ala Ser Val Glu Gly Pro
435 440 445
att gga cta atg gga agt tca tcc tgc att tcc act caa gga aat ggg 1392
Ile Gly Leu Met Gly Ser Ser Ser Cys Ile Ser Thr Gln Gly Asn Gly
450 455 460
ttg cct gca att atg aca gaa aaa tgt tct gcc ccc aac aac act ctg 1440
Leu Pro Ala Ile Met Thr Glu Lys Cys Ser Ala Pro Asn Asn Thr Leu
465 470 475 480
tgg agc aca gtc tcc tct gga cag ctg cag ttg ggc act aga gtt ttt 1488
Trp Ser Thr Val Ser Ser Gly Gln Leu Gln Leu Gly Thr Arg Val Phe
485 490 495
gat gag gat ggg aaa gag aag tgg atg tgt tta gat ggg agt aga agt 1536
Asp Glu Asp Gly Lys Glu Lys Trp Met Cys Leu Asp Gly Ser Arg Ser
500 505 510
cca ttg att aca aca act gaa tgc atc tgc atc act gac tct cac tgc 1584
Pro Leu Ile Thr Thr Thr Glu Cys Ile Cys Ile Thr Asp Ser His Cys
515 520 525
tat cca aat cag aat cca gaa aag cag tgg ttt aaa gtc ata aca acc 1632
Tyr Pro Asn Gln Asn Pro Glu Lys Gln Trp Phe Lys Val Ile Thr Thr
530 535 540
aac aaa caa ttg ttg cat caa ctt tga 1659
Asn Lys Gln Leu Leu His Gln Leu
545 550
<210> 2
<211> 552
<212> PRT
<213> Artificial sequence
<220>
<223> Synthetic Construct
<400> 2
Met Thr Ala Thr Ala Met Thr Thr Ala Ala Leu Arg Leu Leu Ile Ala
1 5 10 15
Leu Leu Leu Val Ala Ala Pro Ala Glu Cys Leu Pro Leu Arg Ala Arg
20 25 30
Gly Arg Trp Ile Val Asp Glu Ala Thr Gly Leu Arg Val Lys Leu Ala
35 40 45
Cys Val Asn Trp Pro Gly His Leu Glu Pro Gly Leu Pro Glu Gly Leu
50 55 60
Asn Arg Leu Pro Val Thr Thr Ile Ala His Thr Ile Ser Ser Leu Gly
65 70 75 80
Phe Asn Cys Val Arg Leu Thr Tyr Ser Ile Gln Met Val Thr Glu Lys
85 90 95
Ser Tyr Thr Glu Ala Thr Val Gly Gln Thr Phe Ala Gln Leu Asn Leu
100 105 110
Thr Glu Pro Ala Ser Gly Ile Glu Gly Asn Asn Pro Gly Phe Leu Gln
115 120 125
Leu Gly His Val Ala Ala Tyr Asp Ser Ile Val Ala Ala Leu Ala Glu
130 135 140
Ala Gly Val Met Val Ile Leu Asp Asn His Val Ser Lys Pro Lys Trp
145 150 155 160
Cys Cys Ala Val Asp Asp Gly Asn Gly Phe Phe Gly Asp Ser Tyr Phe
165 170 175
Asp Pro Arg Leu Trp Gln Arg Gly Leu Gly Leu Met Ala Thr His Phe
180 185 190
Asn Asn Thr Pro Asn Val Val Ala Met Ser Leu Arg Asn Glu Leu Arg
195 200 205
Gly Asn Arg Ser Thr Ser Ala Arg Trp Ser Lys Tyr Met Gln Arg Gly
210 215 220
Ala Ala Thr Val His Glu Ala Asn Pro Asn Val Leu Val Val Leu Ser
225 230 235 240
Gly Leu His Phe Asp Thr Ile Leu Ser Phe Leu Pro Val Leu Pro Val
245 250 255
Thr Leu Pro Phe Lys Glu Lys Ile Val Tyr Glu Gly His Trp Tyr Ser
260 265 270
Phe Gly Val Pro Trp His Asp Gly Leu Pro Asn Asp Ile Cys Leu Asn
275 280 285
Glu Thr Ser Arg Phe Lys Asp Asn Ile Gly Phe Leu Thr Ser Ser Ile
290 295 300
Asn Gly Thr Ala Ala Pro Leu Phe Val Ser Glu Phe Gly Ile Asp Gln
305 310 315 320
Arg Tyr Val Asn Asp Asn Asp Asn Arg Tyr Leu Asn Cys Ile Leu Ala
325 330 335
Phe Leu Ala Glu Glu Asp Val Asp Trp Ala Leu Trp Thr Met Gly Gly
340 345 350
Ser Tyr Asn Tyr Arg Ser Asp Lys Glu Pro Val Gln Asp Phe Glu Glu
355 360 365
Thr Tyr Gly Phe Phe Asn Arg Asp Trp Ser Arg Ile Arg Asn Pro Asp
370 375 380
Phe Ile Ser Arg Leu Lys Glu Ile Gln Gln Pro Ile Gln Asp Pro Tyr
385 390 395 400
Leu Ser Pro Gly Pro Tyr Tyr Gln Ile Ile Tyr His Pro Ala Ser Gly
405 410 415
Leu Cys Val Glu Ser Ser Ile Gly Asn Thr Ile His Leu Gly Ser Cys
420 425 430
Gln Ser Val Arg Ser Arg Trp Asn Tyr Asp Ala Ser Val Glu Gly Pro
435 440 445
Ile Gly Leu Met Gly Ser Ser Ser Cys Ile Ser Thr Gln Gly Asn Gly
450 455 460
Leu Pro Ala Ile Met Thr Glu Lys Cys Ser Ala Pro Asn Asn Thr Leu
465 470 475 480
Trp Ser Thr Val Ser Ser Gly Gln Leu Gln Leu Gly Thr Arg Val Phe
485 490 495
Asp Glu Asp Gly Lys Glu Lys Trp Met Cys Leu Asp Gly Ser Arg Ser
500 505 510
Pro Leu Ile Thr Thr Thr Glu Cys Ile Cys Ile Thr Asp Ser His Cys
515 520 525
Tyr Pro Asn Gln Asn Pro Glu Lys Gln Trp Phe Lys Val Ile Thr Thr
530 535 540
Asn Lys Gln Leu Leu His Gln Leu
545 550
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> artificially synthesized oligonucleotide DNA
<400> 3
gatttcatgr cagcgacag 19
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> artificially synthesized oligonucleotide DNA
<400> 4
catgrcagcg acagsgat 18
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> artificially synthesized oligonucleotide DNA
<400> 5
tcaaagttga tgcaacaatt g 21
Claims (9)
1. A cedar pollen protein is characterized in that:
which is a protein having an amino acid sequence represented by SEQ ID NO. 2.
2. A polynucleotide, wherein:
which encodes a protein having an amino acid sequence represented by SEQ ID NO. 2.
3. A polynucleotide, wherein:
which is a polynucleotide having a base sequence represented by SEQ ID NO. 1.
4. A preventive or therapeutic agent for allergic diseases caused by cedar pollen, comprising the cedar pollen protein according to claim 1 as an active ingredient.
5. A diagnostic agent for allergic diseases caused by cedar pollen, comprising the cedar pollen protein of claim 1 as an active ingredient.
6. Use of the cedar pollen protein according to claim 1 for producing a prophylactic or therapeutic agent for allergic diseases caused by cedar pollen.
7. Use of the cedar pollen protein according to claim 1 for the manufacture of a diagnostic agent for allergic diseases caused by cedar pollen.
8. The cedar pollen protein of claim 1, wherein:
it can be used for preventing or treating allergic diseases caused by cedar pollen.
9. The cedar pollen protein of claim 1, wherein:
it can be used for diagnosing allergic diseases caused by cedar pollen.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016-122318 | 2016-06-21 | ||
| JP2016122318 | 2016-06-21 | ||
| PCT/JP2017/022604 WO2017221910A1 (en) | 2016-06-21 | 2017-06-20 | New cedar pollen protein |
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| CN109312325A CN109312325A (en) | 2019-02-05 |
| CN109312325B true CN109312325B (en) | 2022-04-26 |
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| CN201780036197.6A Active CN109312325B (en) | 2016-06-21 | 2017-06-20 | Novel cedar pollen protein |
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| JP (1) | JP6813577B2 (en) |
| KR (1) | KR102156006B1 (en) |
| CN (1) | CN109312325B (en) |
| TW (1) | TWI762488B (en) |
| WO (1) | WO2017221910A1 (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006217896A (en) * | 2005-02-14 | 2006-08-24 | Japan Science & Technology Agency | New allergen derived from ceder pollen |
| JP2006217830A (en) * | 2005-02-08 | 2006-08-24 | Hiroshima Univ | New protein derived from cedar pollen and gene encoding the protein and their utilization |
| JP2006238861A (en) * | 2005-02-07 | 2006-09-14 | Hiroshima Univ | New allergen derived from japanese cedar pollen and use of the allergen |
| CN102552195A (en) * | 2010-12-10 | 2012-07-11 | 日东电工株式会社 | Sheet-form preparation and method for producing the same |
| CN102753683A (en) * | 2009-10-30 | 2012-10-24 | 日本制纸株式会社 | Protein having cedar pollen immunogenicity, polynucleotide for coding for said protein, and uses of said protein and polynucleotide |
| CN104602698A (en) * | 2012-06-29 | 2015-05-06 | 切尔卡西亚有限公司 | Japanese cedar peptide for preventing or treating allergy |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4102432B2 (en) * | 1992-09-01 | 2008-06-18 | メルク パテント ゲーエムベーハー | Allergenic proteins and peptides derived from cedar pollen |
| JPH06197768A (en) * | 1993-01-07 | 1994-07-19 | Meiji Seika Kaisha Ltd | Cedar pollen antigen and gene coding the same |
| JPH07188289A (en) * | 1993-12-27 | 1995-07-25 | Hayashibara Biochem Lab Inc | Polypeptide, its production and use |
| JP4732040B2 (en) * | 2005-07-11 | 2011-07-27 | 独立行政法人科学技術振興機構 | New allergen derived from Japanese cedar pollen |
-
2017
- 2017-06-20 JP JP2018524100A patent/JP6813577B2/en active Active
- 2017-06-20 KR KR1020187034576A patent/KR102156006B1/en active IP Right Grant
- 2017-06-20 CN CN201780036197.6A patent/CN109312325B/en active Active
- 2017-06-20 WO PCT/JP2017/022604 patent/WO2017221910A1/en active Application Filing
- 2017-06-21 TW TW106120701A patent/TWI762488B/en active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006238861A (en) * | 2005-02-07 | 2006-09-14 | Hiroshima Univ | New allergen derived from japanese cedar pollen and use of the allergen |
| JP2006217830A (en) * | 2005-02-08 | 2006-08-24 | Hiroshima Univ | New protein derived from cedar pollen and gene encoding the protein and their utilization |
| JP2006217896A (en) * | 2005-02-14 | 2006-08-24 | Japan Science & Technology Agency | New allergen derived from ceder pollen |
| CN102753683A (en) * | 2009-10-30 | 2012-10-24 | 日本制纸株式会社 | Protein having cedar pollen immunogenicity, polynucleotide for coding for said protein, and uses of said protein and polynucleotide |
| CN102552195A (en) * | 2010-12-10 | 2012-07-11 | 日东电工株式会社 | Sheet-form preparation and method for producing the same |
| CN104602698A (en) * | 2012-06-29 | 2015-05-06 | 切尔卡西亚有限公司 | Japanese cedar peptide for preventing or treating allergy |
Non-Patent Citations (4)
| Title |
|---|
| Detection of New Antigenic Proteins in Japanese Cedar Pollen;Daichi MATSUMURA et al.;《Biol. Pharm. Bull.》;20061231;第29卷(第6期);第1162-1166页 * |
| Isolation and characterization of native Cry j 3 from Japanese cedar (Cryptomeria japonica) pollen;T. Fujimura et al.;《Allergy》;20081125;第62卷(第5期);第547-553页 * |
| 日本柳杉花粉变应原CJP-6的重组表达和免疫反应性鉴定;***等;《中国免疫学杂志》;20121231;第28卷(第5期);第444-448页 * |
| 柳杉花粉症;许丽娟等;《临床耳鼻咽喉科杂志》;20030831;第17卷(第8期);第502-504页 * |
Also Published As
| Publication number | Publication date |
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| JPWO2017221910A1 (en) | 2019-03-14 |
| KR20180137563A (en) | 2018-12-27 |
| CN109312325A (en) | 2019-02-05 |
| WO2017221910A1 (en) | 2017-12-28 |
| KR102156006B1 (en) | 2020-09-15 |
| TWI762488B (en) | 2022-05-01 |
| JP6813577B2 (en) | 2021-01-13 |
| TW201802111A (en) | 2018-01-16 |
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